Terence Moyana

A comparative immunohistochemical study of jejunoileal and appendiceal carcinoids. Implications for histogenesis and pathogenesis

Background. The purpose of this study was to determine the histogenesis of jejunoileal and appendiceal carcinoids and to ascertain whether this could be useful in further explaining the pathology of these neoplasms.

A genetically engineered single-chain FVTNF molecule possesses the anti-tumor immunoreactivity of FV as well as the cytotoxic activity of tumor necrosis factor

Recombinant DNA techniques were used to clone, construct and express the fused gene FV-TNF in E. coli under control of the strong T7 bacteriophage promoter in the expression vector pT7-7-FV-TNF. The fusion protein FV/TNF in inclusion bodies from the bacteria homogenate was solubilized in the denaturing solution containing 6 mol/l guanidine and 0.3 mol/l DTT and refolded in refolding buffer containing 8 mmol/l GSSG. The FV/TNF was purified by ion exchange chromatography. The yield of FV/TNF was estimated at 10 mg/l. The purified FV/TNF displayed a single band of 42 kD under reducing conditions, whereas it showed three forms including its monomer (40/42 kD), its dimer (84 kD) and its trimer (126 kD) under non-reducing conditions. Our data showed that this fusion protein retained its bifunctional activities well, namely the anti-TAG72 immunoreactivity of the FV portion and the cytotoxic activity of the TNF moiety. Therefore, the FV/TNF fusion protein may prove useful in targeting the biological effect of TNF to tumor cells as well as in stimulating the immune destruction of tumor cells.

Identification of a decapeptide with the binding reactivity for tumor-associated TAG72 antigen from a phage displayed library.

Peptide ligands for tumor‐associated TAG72 antigen were identified by screening a large, diverse decapeptide library expressed on the surface of filamentous phages. Fifty‐eight clones of phages were selected from the eluates after the third round of biopanning and their DNA inserts were sequenced. A dominant decapeptide HYVSIELPDH (14/58) was found with the binding reactivity for TAG72 antigen in the TAG72‐binding ELISA and Western dot blotting. It also showed a preferential binding to colonic adenocarcinomatous cells expressing the TAG72 antigen in the histochemical study. Therefore, this anti‐TAG72 decapeptide may be useful in serving as the starting point with regard to further designing peptidomimetics for potential pharmaceuticals.

Expression of tumor-associated polymorphic epithelial mucin and carcinoembryonic antigen in gastrointestinal carcinoid tumors. Implications for immunodiagnosis and immunotherapy

Background. Gastrointestinal neoplastic epithelium of glandular origin commonly produces N‐linked and O‐linked glycoproteins such as carcinoembryonic antigen (CEA) and tumor‐associated polymorphic epithelial mucin (PEM). Antibodies to these glycoproteins increasingly are being used in immunodiagnosis and/or immunotherapy. Although GI carcinoid tumors have a neuroendocrine immunophenotype and generally have an indolent clinical course compared with their adenocarcinomatous counterparts, they also arise from undifferentiated crypt epithelium. The purpose of this study was to determine whether GI carcinoids similarly expressed epitopes for CEA and PEM.

Production and characterization of a tumor-specific monoclonal antibody ACT19 recognizing an epitope distinctive from sialosyl-Tn on the TAG72 antigen.

Aims A murine monoclonal antibody ACT19 directed at the TAG72 tumor-associated antigen, which was originally defined by the B72.3 antibody, was established. Methods This was done by immunizing mice with the bovine mucin followed by the selection of hybridomas secreting antibodies with the desired specificity. In order to better characterize this antibody, its immunoreactivity was compared to that of the B72.3 antibody. Results The data showed that the ACT19 antibody bound specifically to the TAG72 antigen as the B72.3 antibody did. However, there were some differences between ACT19 and B72.3. Firstly, the immunoreactivity of ACT19 for the bovine mucin was lower than that of B72.3. Secondly, the immunoreactivity of ACT19 for the TAG72 antigen was not inhibited by N-acetylgalactosamine, nor was that of B72.3. Thirdly, ACT19 did not compete the binding reactivity of B72.3 for the TAG72 antigen. This suggests that the epitope defined by ACT19 is different from the sialosyl-Tn epitope recognized by B72.3. Immunoperoxidase staining of various tumors, normal and embryonic tissues for ACT19 was carried out. For the various tumors, only adenocarcinomas from the colon and stomach showed remarkable positivity. All the normal tissues were negative, except for weak positivity involving the zona reticularis of the adrenal cortex, and intestinal goblet cells. Embryonic tissues showed a wide spectrum of positivity with staining of the small and large intestine, stomach and renal tubules. Conclusions The ACT19 antibody appears to be a useful marker for colon and stomach cancers, and this additional anti-TAG72 antibody may be useful in conjunction with the B72.3 antibody in pathology and clinical application.

Antibody-targeted lymphokine-activated killer cells inhibit liver micrometastases in severe combined immunodeficient mice

BACKGROUND & AIMS Animal models for hepatic metastases can facilitate the investigation of lymphokine-activated killer (LAK) cell-based immunotherapy. The aim of this study was to investigate the efficacy of ccM4 antibody-targeted LAK cells in inhibiting hepatic micrometastases. METHODS Hepatic micrometastases were generated after the intrasplenic injection of HM7 colon carcinoma cells. TAG72 expression was detected in these hepatic micrometastases using ccM4 antibody. The ccM4 antibody was conjugated onto LAK cells by treatment with 17.5% polyethylene glycol 8000. After the intrasplenic injection of HM7 cells, severe combined immunodeficient mice were randomized into five groups (i-v) and received either 10(7) ccM4-LAK cells plus 1000 U interleukin 2 (IL-2; group i), LAK cells plus 50 micrograms ccM4 and IL-2 (group ii), LAK cells plus IL-2 (group iii), IL-2 alone (group iv), or only phosphate-buffered saline (group v). RESULTS The ccM4-LAK cells retained cytolytic activity and acquired TAG72-binding reactivity. The results showed that group i had significantly fewer hepatic metastases compared with group ii or group iii (P < 0.05) and even fewer hepatic metastases compared with group iv or group v (P < 0.001). CONCLUSIONS These results show that ccM4 antibody-targeted LAK cells significantly inhibited tumor growth in vivo; thus, they can be potentially useful in treatment of hepatic micrometastases.

Enzyme-linked immunosorbent assay-based selection and optimization of elution buffer for TAG72-affinity chromatography

An enzyme-linked immunosorbent assay (ELISA)-elution assay was developed to screen a large variety of elution buffers for selection of a suitable one for purification of the fusion protein FV/TNF-alpha by affinity chromatography. Various commonly used buffer systems utilizing widely differing conditions such as extreme pH, denaturants, chaotropic ions and polarity reducing reagents were investigated. Ammonia solution (1 M, pH 11.5) proved to exert the most suitable influence on dissociation of the FV/TNF-alpha/TAG72 complex while having a minimal protein denaturing effect on FV/TNF-alpha. The total yield of purified FV/TNF-alpha using the TAG72-affinity column with this elution system was 300-fold higher than that using the common elution buffer, 0.1 M glycine, 0.5 M NaCl, pH 2.7. Our study indicates that the ELISA-elution assay will be most useful in the selection of suitable elution buffers for affinity chromatography.

Characterization of anti-tumor immunity derived from the inoculation of myeloma cells secreting the fusion protein RM4/IFN-τ

Our previous study showed that the injection of mouse myeloma VKCK/RM4-IFN-tau cells secreting the fusion protein RM4/IFN-tau to syngeneic BALB/c mice resulted in tumor regression in 70% of mice after tumor inoculation. In this study, the VKCK/RM4-IFN-tau cell line was used to characterize the protective immunity subsequent to tumor inoculation. Our histologic findings demonstrated that, in the primary response to VKCK/RM4-IFN-tau inoculation, tumor regression is associated with macrophage infiltration. This macrophage-dominated regression further leads to a protective immunity against the 2nd challenge of parental VKCK tumor cells. FACS analysis and chromium release assays showed that the majority of T lymphocytes that mediated this anti-tumor immunity were CD8+ cytotoxic T lymphocytes (CTLs). Our animal studies further showed that the VKCK/RM4-IFN-tau cells were able to grow as aggressively as the parental VKCK cells in T lymphocyte deficient nude mice. The protective immunity started 7 days, became complete 10 days following and lasted up to at least 12 months subsequent to the tumor inoculation. The adoptive transfer of T lymphocyte-enriched spleen cells or CTLs also conferred significant protection against tumor growth of parental VKCK cells (p < 0.01). These data thus support the notion that genetically engineered tumor cells secreting IFN-tau may have potential use as tumor vaccines in preventing the development of tumor recurrence and/or metastases following the surgical removal of the primary tumors.